title: Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy.
abstract: BACKGROUND
  The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients.
  
  
  METHODS
  We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay) as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.
  
  
  RESULTS
  In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154-162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (approximately 1010 cells).
  
  
  CONCLUSIONS
  This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.
authors.affiliation: Surgery Branch, National Cancer Institute, Bethesda, MD, USA. udai_kammula@nih.gov
authors.forename: Udai S
authors.initials: US
authors.lastname: Kammula
authors.forename: Oscar K
authors.initials: OK
authors.lastname: Serrano
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology; D001681:Biological Assay; D002460:Cell Line; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000276:immunology; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins